Compositions for treating cystic fibrosis
a technology for cystic fibrosis and compositions, applied in the field of compositions for treating cystic fibrosis, can solve the problems of increasing the frequency, duration and severity of lung infections, affecting the survival rate of patients, so as to reduce or eliminate the symptoms of cystic fibrosis, the effect of reducing the ige level
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example 1
[0126]This Example summarizes results of preclinical studies in which pseudotyped AAV2 / 5 vectors were utilized to combine high dose airway delivery with a serotype preferential to the airway surface.
[0127]AAV2 / 5 pseudotyped (rep2 cap5) vectors (both AAV2 / 5-GFP and AAV2 / 5-CFTR) were administered at doses up to 1×1014 DRP / lung in rhesus macaques by endobronchial delivery with a PennCentury Microsprayer™. Gene transfer and expression of the GFP transgene was detected by real time PCR and RT-PCR using the Light cycler (Roche). GFP transfer was 100× greater in these AAV2 / 5 studies at 1×106 copies / μg of DNA as compared with that achievable with AAV2. GFP RNA expression was present and quantified up to 2×105 copies / μg of cDNA. In comparison, RNA expression was only qualitatively detected by nested RT-PCR after AAV2 dosing.
[0128]FIG. 13 is a graphical representation of the results, showing copies of DNA in each lung region measured in monkeys infected with either single or repeated dosing o...
example 2
[0131]Recombinant adeno-associated viral vectors lack vector-induced inflammation and have the ability to integrate into a subject's genome. AAV-serotype 2 (AAV2) has been the serotype most utilized in clinical trials and feasibility has been confirmed with successful gene transfer. This Example describes the use of pseudotyped vectors, which use capsid proteins of other AAV serotypes.
[0132]Methods: AAV2 / 5 pseudotyped vectors were generated using the AAV2 ITRs with the AAV5 capsid proteins expressing 1) a truncated CFTR insert (Δ268) capable of reconstituting physiologic function or 2) an reporter insert, GFP, whose expression was driven by the chicken beta actin promoter (CBA).
[0133]AAV2 / 5 pseudotyped vectors with either the CFTR insert or the reporter insert, as a control vector, were administered at doses up to 1×1014 DRP / lung in nonhuman primates by aerosolized bronchoscopic delivery with a Penn Century Microsprayer™. Animals were closely observed and sacrificed at day 21, at wh...
example 3
[0136]Juvenile rhesus macaques are obtained from the Johns Hopkins University breeding colony and housed according to Animal Care and Use Committee guidelines. All macaques are tested for simian immunodeficiency virus and Herpesvirus simiae (Herpes B), and are judged to healthy by the veterinarian before any procedure.
[0137]Radiolabeled saline and rAAV-GFP vectors are admixed with radiolabel. Three hundred μCi of the radioisotope 99mtechnetium (99 mTc) (Syncor, Baltimore, Md.) are chelated to diethylene-triamine penta-acetic acid (DTPA) and admixed with 3 ml of normal saline or with 3 ml of saline and rAAV-GFP vector. To ensure that the addition of the isotope and DTPA did not alter the viability of the vector, infectivity of Human Embryonic Kidney (HEK) cells treated with rAAV-GFP alone was compared to infectivity in cells that were treated with vector admixed with 99m Tc-DTPA-saline.
[0138]Cells are analyzed by fluorescence activated cell sorting (FACS) and by fluorescence microsco...
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